The RNA Viruses Section entered into a CRADA with MedImmune in 2006 to develop live attenuated vaccines against human respiratory syncytial virus (RSV), human metapneumovirus (HMPV), and human parainfluena viruses (HPIV3) serotypes 1, 2, and 3. These vaccines are intended for intranasal administration to infants and young children. The first priority is to develop a bivalent vaccine against RSV-A and HPIV3, the two more prevalent and important viruses in this group. It is expected that immunization with RSV-A will provide substantial cross-protection against RSV-B. Secondary objectives are to develop live-attenuated vaccines against RSV-B, HMPV, and HPIV1 and 2. Vaccine virus candidates are generated from cDNA by reverse genetics, providing well-defined vaccines. Stepwise clinical evaluation begins in adults (who are seropositive for these common viruses), followed by seropositive older children (typically 15-59 months of age), followed by seronegative younger children and infants (typically 6 -59 months of age). In some cases, viruses are evaluated in young infants, typically 1-3 months of age. The following is a summary of the studies to date. RSV-A vaccine: Under the CRADA, Medimmune is conducting a Phase 1/2a immunogenicity study in young infants with a recombinant rRSV (called rA2cp248/404/1030delSH or Medi-559) that contains a set of five independent attenuating mutations. In a previous phase 1 study by LID, this virus was well-tolerated and immunogenic in 1-2 month old infants. The purpose of the present study is to get further data on safety and immunogenicity. LID also has initiated a clinical study of a live attenuated rRSV bearing deletion of the M2-2 coding sequence (Medi delM2-2). This virus exhibits up-regulated gene expression that may increase its immunogenicity. Manufacturing is in progress for a vaccine candidate containing a deletion in the NS1 gene. Other candidates are in pre-clinical development. HPIV3 vaccine: There are two HPIV3 vaccine candidates in ongoing clinical trials. (i) The first is rB/HPIV3, which is recombinant chimera consisting of bovine PIV3 (BPIV3) in which the F and HN protective antigen genes have been replaced by those of HPIV3. The BPIV3 backbone provides a host-range attenuation phenotype to which each of the genes contributes. In unscreened adults (N =15) and seropositive older children (N = 15, with a vaccine to placebo ratio of 2:1), there was minimal shedding and no difference in illness between vaccinees and placebo recipients. In seronegative children of 6 to 36 months of age (N = 21, with a vaccine to placebo ratio of 2:1), the vaccine infected all recipients, replicated to low level (mean peak titer 2.8 log10 pfu/ml), was well-tolerated, and induced a &#8805;4-fold rise in serum antibodies in 93% of the recipients. This, rB/HPIV3 is a promising vaccine candidate. (ii) The second HPIV3 candidate is rHPIV3cp45. This is a recombinant version of a biologically derived cold-passaged (cp) virus that previously had been found by LID and collaborators to exhibit satisfactory infectivity, safety, immunogenicity, and lack of transmissibility in seronegative infants and children. LID re-derived this virus from cDNA to provide a known pedigree for safety reasons, and the attenuating mutations were identified as described in previous reports. The vaccine was evaluated in a 2-dose, double-blind, placebo controlled trial in 6-12 month old serologically-unscreened infants. 12 of the 15 infants receiving the first dose shed vaccine virus at low-to-moderate titers, and there was no difference in illness between vaccine and placebo recipients. 10 of 15 vaccinated children and none of the 8 placebo recipients had a 4-fold or greater rise in virus-specific serum antibody titer. Of the 5 vaccinated infants who did not respond serologically, each was found to have pre-existing serum antibodies to HPIV3 Following the second dose, given after a mean interval of 6 weeks, only 1 of 15 vaccinees shed vaccine virus;this individual was asymptomatic. 13% of recipients of the second dose had respiratory or febrile illness, compared to 63% of the second dose placebo group. None of the vaccinees or placebo recipients had a 4-fold rise in virus-specific serum antibody titer following the second dose. This indicates that the vaccine indeed induced protection against HPIV3 infection, and that a longer interval between doses may be needed to obtain a boosting effect. This protocol was amended to include screening to ensure that the infants are HPIV3-seronegative prior to immunzation, and the age group was expanded to include infants and children ages 6 to 36 months. In addition, nasal washes for viral assay were collected only in the case of respiratory or febrile illness. 21 additional subjects were enrolled, receiving vaccine or placebo in a 2:1 ratio in a double-blinded fashion. The incidence of respiratory or febrile illness was the same in the vaccine and placebo recipients. 4-fold or greater rises in HPIV3-specific serum antibody were observed in 79% and 14% of vaccinees following the first and second dose, respectively. 86% of vaccinees had a 4-fold or greater rise following either dose. In general, this study indicated that the cDNA-derived vaccine material was equivalent to its biologically-derived parent, and thus represents a promising vaccine candidate that should be evaluated further. Under a separate protocol, the effect of increasing the interval between the 2 doses to 6 months is presently being evaluated in companion protocols through Seattle Children's Hospital and the International Maternal Pediatric Adolescent AIDS Clinical Trials (IMPAACT) Group. A total of 40 children have been enrolled. This study should provide information that will help determine the dosing schedule for a phase 2b study. HPIV1 vaccine: We previously used reverse genetics to generate a live attenuated HPIV1 vaccine candidate called rHPIV1-C(R84G/del170)HN(T553A)L(Y942A) that is attenuated by defined mutations including ones that have been engineered for genetic stability. This virus was immunogenic and protective in African green monkeys (AGMs). When given to 35 unscreened healthy adult volunteers, only 4 individuals were infected and shedding was minimal and with no illness, which is the desired safety profile for an attenuated virus. When evaluated in seropositive children 15 to 59 months of age (double-blind, 10 vaccine recipients and 5 placebo), none of the vaccinees were detectably infected, consistent with a highly attenuated nature. The vaccine presently is being evaluated in seronegative children 6 to 59 months of age (N = 30, with a vaccine to placebo ratio of 2:1). Results are incomplete but indicate that vaccine virus infectivity and shedding were low. HPIV2 vaccine: We previously used reverse genetics to generate a live attenuated intranasal HPIV2 vaccine called rHPIV2-V94(15C)/948L/1724 that, like the HPIV1 candidate, included stabilized mutations. This virus was immunogenic and protective in AGMs. When given to 15 seropositive adult volunteers, only 4 were infected and vaccine virus was shed in minimal amounts. The virus is presently being evaluated in seropositive children 15 to 59 months of age (N = 15, with a vaccine to placebo ratio of 2:1). HMPV vaccine: LID has initiated the first HMPV vaccine clinical study to be reported. This involves a live-attenuated HMPV vaccine virus (rHMPV-Pa) in which the HMPV P gene was replaced by that of avian MPV, thus conferring a host range attenuation phenotype. This virus has been administered to 15 healthy adults, with the result that there was no detectable HMPV infection or shedding, consistent with this being a highly attenuated virus.